Recently we described a missense mutation that impairs but does not destroy MLH1 function (referred to as hypomorphic), MLH1 D132H. MLH1 D132H reduces MLH1 activity by ~80%, but is not a classic null mutation causing 100% loss of function like those typically observed in HNPCC. As expected from a hypomorphic mutation, MLH1 D132H causes CRC typically at later ages and in fewer family members than classic mutations causing HNPCC. Unexpectedly, MLH1 D132H CRCs do not have MSI (referred to as Microsatellite Stable or MSS) when tested with the conventional assay used for detecting MLH1 mutations in HNPCC. A significant limitation to CRC prevention in patients with these missense substitutions has been the difficulty interpreting benign variants from deleterious mutations with the high degree of confidence required for clinical diagnosis. The conventional MSI assay is useful to help distinguish benign from deleterious changes. However, it is not a very sensitive test, and does not correctly classify hypomorphic mutations like MLH1 D132H. Single-Molecule MSI is a more sensitive assay than the conventional MSI test used in Clinical Diagnostic Laboratories. We hypothesize that Single-Molecule MSI will be an effective functional assay to identify hypomorphic MLH1/MSH2 mutations, and distinguish them from benign variants. We therefore propose the following Specific Aims: SPECIFIC AIM 1: To Determine the Sensitivity of Single-Molecule MSI To Detect Hypomorphic MSI in CRCs from Patients Carrying the MLH1 D132H Hypomorphic Mutation. SPECIFIC AIM 2: To Test Single-Molecule MSI as a Validation Assay for Additional Predicted MLH1/MSH2 deleterious alleles in MSS CRCs. [unreadable] [unreadable] [unreadable]